Leukaemia inhibitory factor from livestock species and use thereof to enhance implantation and development of embryonic cells

ABSTRACT

The present invention relates generally to the isolation of leukaemia inhibitory factor (LIF) genes from livestock species, the expression of said genes in recombinant vectors and the isolation of the recombinant LIF molecules and the use of livestock species LIF to enhance the in vitro development of an embryo to the implantation stage.

The present invention relates generally to leukaemia inhibitory factor(LIF) from livestock species. More particularly, the present inventionrelates to the identification, cloning and structural characterisationof genes encoding LIF from livestock species. The present invention alsorelates to the use of LIF from livestock species in the enhancement ofdevelopment of mammalian embryos and in maintaining ES cell lines invitro.

LIF is a protein that has previously been cloned, produced and purified,in large quantities in purified recombinant form from both E. coli andyeast cells (International Patent Application No. PCT/AU88/00093.) LIFhas been defined as a factor, the properties of which include:

1. the ability to suppress the proliferation of myeloid leukaemic cellssuch as M1 cells, with associated differentiation of the leukaemiccells; and

2. the ability to compete with a molecule having the defined sequence ofmurine LIF or human LIF (defined in International Patent Application No.PCT/AU88/00093) for binding to specific cellular receptors on M1 cellsor murine or human macrophages. In addition to the biological propertiespreviously disclosed for murine and human LIF, LIF has been found tohave the following additional properties:

(a) it maintains in vitro in the absence of feeder cells, thepluripotential phenotype of murine embryonic stem (ES) cell lines: D3and EK-cs4 (derived from strain 129/SV blastocysts) and CBL63 and MD5(derived from C57BL/6 blastocysts) as disclosed in InternationalApplication No. PCT/AU89/00330;

(b) it allows the aforementioned ES cell lines, after passage in vitroin the presence of LIF, to contribute to the tissues of chimaeric micewhen re-introduced into the embryonic environment;

(c) it demonstrates selective binding to high affinity receptors onmurine ES (EK-cs1 and D3) and embryonic carcinoma (EC) (PCC3-3A, F9,PC13, P19 and MG2) cells; and

(d) specific binding of ¹²⁵ I-LIF to high affinity receptors is not incompetition with insulin, IGF-I, IGF-II, acidic and basic FGF, TGFβ,TNFα, TNFβ, NGF, PDGF, EGF, IL-1, IL-2, IL-4, GM-CSF, G-CSF, Multi-CSFnor erythropoietin, but is in competition with murine and human LIF.

Accordingly, LIF is a potent hormone having utility in the general areaof in vitro embryology, such as in maintaining ES cell lines andincreasing the efficiency of embryo transfer and thus has importantapplications in the livestock industry. This is particularly apparent inthe use of ES cells to provide a route for the generation of transgenicanimals.

A major difficulty associated with present in vitro fertilisation (IVF)and embryo transfer (ET) programmes, particularly in humans, is the lowsuccess rate "achieved" on implantation of fertilised embryos.Currently, in human IVF programmes, the implantation rate may be as lowas 10%, leading to the present practice of using up to four fertilisedembryos in each treatment which, in turn, leads occasionally to multiplebirths. Accordingly, there is a need to improve the implantation rate inhuman IVF programmes. Similarly, in IVF and ET treatments in domesticanimals such as sheep, cattle, pigs and goats, it is highly desirablefor economic reasons to have as high as implantation rate as possible soas to reduce the numbers of fertilised embryos lost and unsuccessfultreatment procedures performed.

In the development of a mammalian embryo, the fertilised egg passesthrough a number of stages including the morula and the blastocystsstages. In the blastocyst stage, the cells form an outer cell layerknown as the trophectoderm (which is the precursor of the placenta) aswell as an inner cells mass (from which the whole of the embryo properis derived). The blastocyst is surrounded by the zona pellucida, whichis subsequently lost when the blastocyst "hatches". The cells of thetrophectoderm are then able to come into close contact with the wall ofthe uterus in the implantation stage. Prior to formation of the embryoproper by the inner cell mass by gastrulation, the whole cell mass maybe referred to as "pre-embryo".

Although LIF from one species may be effective, for example inmaintaining ES cell lines from a different or heterologous species, itmay be preferable to develop homologous systems employing LIF and EScell lines derived from the same species. It has now been found, inaccordance with the present invention, that murine LIF DNA can be usedto identify the LIF gene from a wide range of mammalian genomes and toclone the gene encoding LIF from livestock species such as pigs andsheep and hence, provide a source of LIF for use in the development of avariety of in vitro embryogenic procedures, such as ES cell lines andembryo transfer in livestock species.

Furthermore, it has also been found that when LIF is included in an invitro embryo culture medium, the hatching process is enhanced leading toan increased number of embryos completing the development stage byundergoing developmental changes associated with implantation. As aresult, the implantation rate for IVF and ET programmes can besignificantly improved by the use of LIF in the in vitro embryo culturemedium.

Accordingly, one aspect of the present invention relates to the LIF genefrom any livestock species which can be detected by cross-hybridizationwith a nucleotide probe to murine LIF. That is, a first nucleic acidmolecule, encoding a livestock species leukaemia inhibitory factor,comprising a nucleotide sequence capable of hybridizing to a secondnucleic acid molecule which encodes murine leukaemia inhibitory factoror part thereof.

A "nucleotide probe" as used herein means a DNA or RNA sequence or anycombination thereof capable of detecting complementary sequences byhybridization techniques such as, but not limited to, Southern orNorthern blotting or colony hybridization. The probe may comprise asmall number of nucleotides (eg. 6-20) or may be the entire gene or partor parts of a gene. The probe may be labelled with a detectable signal(eg. radioactive isotope).

By "nucleic acid" is meant a polymer of four or more nucleotides inwhich the 3' position of one nucleotide sugar is linked to the 5'position of the next nucleotide by a phosphodiester bridge. The nucleicacid contemplated herein may be linear or circular, single or doublestranded DNA or RNA.

"Livestock species" is used herein in its most general senseencompassing, but not limited to, sheep, cows, pigs, horses, donkeys andthe like. Even more preferably, the livestock species is sheep or pig.

By "hybridizing" is meant the ability to form a double stranded thirdnucleic acid by the formation of base pairs between single strands ofthe first and second nucleic acid under appropriate conditions ofstringency. The stringency conditions employed will depend on therelative homology between the relevant strands of the first and secondnucleic acid molecules. Convenient conditions for stringency can befound in Maniatis et al. (1982) or by reference to the non-limitingexamples of the present specification.

Accordingly, where the nucleic acids are double stranded molecules, thepresent invention relates to a first nucleic acid encoding part or partsof livestock species leukaemia inhibitory factor comprising on onestrand thereof a nucleotide sequence capable of being hybridized to by astrand of a second nucleic acid encoding part or parts of murine LIF.

Although the present invention is exemplified by the second nucleic acidencoding murine LIF or parts thereof, it is possible that a differentnucleic acid encoding non-murine LIF but which is capable of hybridizingto murine-LIF nucleic acid could be used. The use of non-murineLIF-encoding second nucleic acid is, therefore, still encompassed by thepresent invention provided said non-murine LIF-encoding nucleic acid iscapable of being hybridized to by the said second nucleic acid encodingmurine LIF or parts thereof.

The present invention extends to nucleic acids encoding full length LIFmolecules or to part or parts of LIF molecules. Accordingly, the nucleicacids may represent the full coding sequence of mammalian LIF or carrysingle or multiple nucleotide additions, deletions and/or substitutionsor may represent just a portion of the LIF molecule, for example anN-terminal or C-terminal portion. Accordingly, "parts" of a LIF moleculeincludes any one or more contiguous series of amino acids containedwithin a LIF molecule and further includes natural, chemical and/orrecombinant derivatives.

Another aspect of the present invention relates to a recombinant DNAmolecule containing the nucleotide sequence encoding LIF from alivestock species or substantially similar analogues thereof, eithercompletely or in part, in a form in which said nucleotide sequence isable to direct the synthesis and production of said LIF, eithercompletely or in part. This aspect of the invention also extends tocloning vectors such as plasmids and expression vectors and host cellshaving such recombinant DNA molecules inserted therein. Furthermore, theinvention also extends to synthetic livestock LIF, either complete or inpart, or substantially similar analogues thereof, produced by expressionof such recombinant DNA molecules.

Accordingly, this aspect of the present invention relates to recombinantDNA or RNA molecules comprising the first nucleic acid defined aboveoperably linked to one or more regulatory regions such that in theappropriate host and under the requisite conditions, the first nucleicacid will be transcribed and translated into a recombinant LIF productor derivative or part thereof. The recombinant molecule will furthercomprise a replication region appropriate for the intended host or maycomprise more than one replication region if more than one host is used.Vectors and suitable hosts are known to those skilled in the art and arediscussed in the non-limited examples herein, in PCT/AU88/00093 and inManiatis et al. (1982).

The present invention, therefore, extends to recombinant livestock LIF,and preferably, but not limited to, ovine and porcine LIF or derivativesor parts thereof. Such derivatives or parts thereof are discussed abovebut include single or multiple amino acid substitutions, deletionsand/or additions to or in the natural or synthetic livestock LIFmolecule. Conditions for preparing recombinant LIF are disclosed inPCT/AU88/00093 although variations and/or modifications to theseconditions may vary depending on the host cell used. Any such variationsand/or modifications are within the scope of the subject invention. Thehost cells may be eukaryotic (e.g. yeast, mammalian, plant etc.) cellsor prokaryotic (e.g. Escherichia coli, Bacillus sp, Pseudomonks spetc.).

Yet another aspect of the present invention provides a source ofrecombinant livestock LIF for use in in vitro embryology. Accordingly,the present invention contemplates a method for maintaining ES celllines in in vitro culture while retaining a pluripotential phenotypewhich method comprises contacting said ES cell lines with an ES cellline maintaining effective amount of livestock species LIF forsufficient time and under appropriate conditions.

Still yet another aspect of the present invention relates to a methodfor enhancing the in vitro development of a mammalian embryo to theimplantation stage, which method comprises the step of culturing theembryo in vitro in a culture medium containing an effective amount ofmammalian LIF. Preferably, pre-embryos are allowed to develop to thestage of formation of the blastocyst (post-hatching embryos) before LIFis included in the culture medium as LIF has been found to enhance thehatching process leading to an increased number of embryos completingthe developmental stage. As is demonstrated below, however, theinclusion of LIF in the culture medium prior to the formation of theblastocyst, or both prior to and following blastocyst formation, alsoincreases the number of pre-embryos completing the developmental stageby undergoing development changes associated with implantation. As aresult, the implantation rate for IVF and ET programmes can besignificantly improved by use of LIF in the in vitro culture medium.

"Mammalian embryos" is used in its broadest sense encompassing human,ruminant and other livestock animals. It will be appreciated that whilethe subject invention is exemplified herein by the development of murineembryos in vitro, the present invention extends to the use of LIF in thedevelopment of embryos of other species including humans, ruminants andanimals such as sheep, cattle, horses, donkeys, goats and the like.

The present invention, also extends to a method for in vitrofertilisation and subsequent implantation of a mammalian embryo which ischaracterised in that the embryo is cultured in vitro in a culturemedium containing an effective amount of mammalian LIF prior toimplantation.

"Mammalian LIF" encompasses human, murine, ruminant and other orlivestock LIF such as from sheep, pigs, cows, goats, donkeys and horsesand the like.

In the figures:

FIG. 1 relates to Example 1. The identification of LIF gene homologuesin DNA from a variety of mammalian species by cross-hybridization with amurine cDNA probe.

FIG. 2 shows the nucleotide sequence of the porcine LIF gene. ThemRNA-synonymous strand of 2 portions of the porcine LIF gene amountingto 2.07 kbp of sequence derived from clone λPGLIF-E2, spanning the twoexons encoding the mature protein of the porcine LIF gene are listed 5'to 3' using the single letter code according to standard practice, whereA refers to deoxyadenosine-5'-phosphate, C refers todeoxycytidine-5'-phosphate, G refers to deoxyguanosine-5'-phosphate andT refers to deoxythyjidine-5'-phosphate. The amino acid sequence encodedby the two exons of the porcine LIF gene defined by homology with themurine, human and ovine cDNA and gene sequences (InternationalApplication No. PCT/AU88/00093) is listed above the gene sequence, whereALA is Alanine, ARG is Arginine, Asn is Asparagine, ASP is Asparticacid, CYC is Cystein, GLN is Glutamine, GLU is Glutamic acid, GLY isGlycine, HIS is Histidine, ILE is Isoleucine, PHE is Phenylalanine, PROis proline, SER is Serine, THR is Threonine, TRP is Tryptophan, TYR isTyrosine, and VAL is Valine.

FIG. 3 shows the nucleotide sequence of the ovine LIF gene. ThemRNA-synonymous strand of three portions of the ovine LIF gene amountingto .sup.˜ 1.5 kbp of sequence derived from clone λOGLIFR2, spanning thethree protein coding regions of the ovine LIF gene are listed 5' to 3'using the single letter code according to standard practice, where Arefers to deoxyadenosine-5'-phosphate, C refers todeoxycytidine-5'-phosphate, G refers to deoxyguanosine-5'-phosphate andT refers to deoxythymidine-5'-phosphate. The amino acid sequence encodedby the three exons of the ovine LIF gene defined by homology with themurine and human cDNA and gene sequences (International Application No.PCT/AU88/00093) is listed above the gene sequence, where ALA is Alanine,ARG is Arginine, ASN is Asparagine, ASP is Aspartic acid, CYS isCysteine, GLN is Clutamine, GLU is Glutamic acid, GLY is Glycine, HIS isHistidine, ILE is Isoleucine, PHE is Phenylalanine, PRO is Proline, SERis Serine, THR is Threonine, TRP is Tryptophan, TYR is Tyrosine, and VALis Valine.

FIG. 4 shows the amino acid sequence of porcine LIF and comparison withmurine, human and ovine LIF. The amino acid sequence of murine LIF (M)as determined by direct amino acid sequencing and nucleotide sequenceanalysis of LIF encoding cDNAs (PCT/AU88/00093) is listed on the topline, the corresponding human and ovine amino acid sequences (H and O)determined by nucleotide sequence analysis of the human and ovine LIFgenes (PCT/AU88/00093) in the middle, and the corresponding sequence ofporcine LIF (P) on the bottom line. The N-terminal residue of maturemurine LIF, determined by direct amino acid sequencing, is designated+1. Identities between murine and human, between human and ovine orbetween ovine and porcine LIF are indicated by asterisks andconservative substitutions (Arg/Lys; Glu/Asp; Ser/Thr; Ile/Leu/Val) bydashes.

EXAMPLE 1 Identification of mammalian LIF genes by cross-hybridizationwith a murine LIF cDNA probe

A method has been previously disclosed (PCT/AU88/00093) for using aradioactively labelled fragment of mouse LIF cDNA as a hybridizationprobe to detect the human LIF gene on Southern blots. FIG. 1demonstrates that similar conditions can be used to detect presumptiveLIF gene homologues in a variety of mammalian DNAs, including sheep,pig, cow, guinea pig, dog, monkey, human and rat. Note that in eachspecies, using this probe, only a unique gene is detected, with noevidence for reiterated sequences. Note also that the intensity ofhybridization of the presumptive LIF gene homologues is less than thatof the murine probe to rodent DNA, implying a lower degree of homology.

Each track on the gel contains 10 μg of genomic DNA from each of theindicated species, digested to completion with the restrictionendonuclease BamHI. After electrophoresis through a 0.8% w/v agarose geland transfer to nitrocellulose using standard conditions, theimmobilized DNA was hybridized with a fragment of murine LIF cDNA fromclone pLIF7-2b (PCT/AU88/00093) ³² p-labelled by nick-translation to aspecific activity of .sup.˜ 2×10⁸ cpm/μg. The filter was prehybridizedand hybridized at 65° C. in 0.9M NaCl, 0.09M Sodium citrate (6×SSC),0.2% w/v Ficoll, 0.2% w/v polyvinylpyrollidine, 0.2% w/v bovineserumalbimun, 50 μg/ml E.coli tRNA, 0.1 mM ATP and 2 mM sodiumpyrophosphate. During hybridization, 0.1% w/v SDS was included and theprobe was included at .sup.˜ 2×10⁷ cpm/ml. After hybridization at 65° C.for 16 hours, the filter was extensively washed in 2×SSC, 0.1% w/v SDSat 65° C. and then autoradiographed using a Kodak XAR5 film and 2screens at -70° C.

EXAMPLE 2 Isolation of the porcine LIF gene

A library of porcine genomic DNA, partially digested with Sau 3A, wasscreened for LIF gene-containing clones by hybridization with both amurine LIF cDNA and a portion of the human LIF gene as probes. Themurine LIF cDNA fragment used as a probe corresponded to the LIF codingregion and was derived from clone pLIFmutl; the human gene fragment usedas a probe corresponded to the 3kbp BamH1 fragment spanning the humanLIF gene and was derived from clone pHGLIFBaml (PCT/AU88/00093).Conditions of hybridization were as previously disclosed(PCT/AU88/00093). Briefly, phage plaques representing the genomiclibrary were grown at a density of 50,000 plaques per 10 cm petri dishand transferred to nitrocellulose as described in Maniatis et al.(1982). Four nitrocellulose filters were prepared from each dish. Priorto hybridization, filters were incubated for several hours at 65° C. in6×SSC (SSC=0.15M NaCl, 0.015M sodium citrate), 0.2% w/v Ficoll; 0.2% w/vpolyvinylpyrollidine; 0.2% w/v bovine serum albumin, 2 mM sodiumpyrophosphate, 1 mM ATP, 50 μg/ml E. coli tRNA 0.1% w/v SDS at 65° C.for 16-18 hours. The murine LIF cDNA and human LIF genomic DNA fragmentswere each radioactively labelled by nick-translation using [α-³² P] dATPto a specific activity of .sup.˜ 2×10⁸ cpm/μg or by random priming to aspecific activity of .sup.˜ 10⁹ cpm/μg and were included in thehybridization at a concentration of .sup.˜ 2×10⁶ cpm/ml. For each petridish, 2 nitrocellulose filters were hybridized with the murine probe andtwo with the human probe. After hybridization, filters were extensivelywashed in 6×SSC, 0.1% w/v SDS at 65° C. and then autoradiographed.Plaques positive on quadruplicate filters were picked and rescreened atlower density, as before. The use of two different probes simultaneouslyreduced the chance of identifying clones containing short sequencesegments displaying fortuitous to one or other of the probes. Of theclones originally identified, one (λPGLIF-E2) was purified. DNA fromthis λ clone was digested with a series of restriction endonucleases(including SalI which liberates the entire segment of cloned genomicDNA). After digestion of the recombinant phage DNAs and resolution byelectrophoresis on agarose gels, the DNA was transferred tonitrocellulose and hybridized with the mouse LIF cDNA probe (under theconditions outlined above) to reveal the fragments containing the LIFgene. Even after washes of higher stringency (0.2×SSC, 65° C.) theporcine DNA still displayed strong hybridization with the murine probe.A 2.4 kbp BamHI fragment hybridizing to the murine cDNA probe andcorresponding in size to that identified in Southern blots of porcinegenomic DNA was identified and subcloned into the plasmid vector pUC12,giving rise to clone pPLIFBaml.

EXAMPLE 3 Isolation of the Ovine LIF gene

A library of ovine genomic DNA, partially digested with Sau 3A andligated with the lambda phage cloning vector EMBL 3A, was screened forLIF gene-containing clones by hybridization with both a murine LIF cDNAand a portion of the human LIF gene as probes. The murine LIF cDNAfragment used as a probe corresponded to the 3 kbp BAMHI fragmentspanning the human LIF gene and was derived from clone pHGLIFBaml(PCT/AU88/00093). Conditions of hybridization were as disclosed inPCT/AU88/00093 and Example 2.

Of the 8 clones originally identified, one (λOGLIFR2) was purified. DNAfrom this λ clone was digested with a series of restrictionendonucleases (including SalI which liberates the entire segment ofcloned genomic DNA). After digestion of the recombinant phage DNAs andresolution by electrophoresis on agarose gels, the DNA was transferredto nitrocellulose and hybridized with the mouse LIF cDNA probe (underthe conditions outlined above) to reveal the fragments containing theLIF gene. Even after washes of higher stringency (0.2×SSC, 65° C.) theovine DNA still displayed strong hybridization with the murine probe. A.sup.˜ 3 kbp BamHI fragment hybridizing to the murine cDNA probe wasidentified and subcloned into the plasmid vector pEMBL8+, giving rise toclone pOGLIFBaml.

EXAMPLE 4 Determination of nucleotide and amino acid sequences of theporcine and ovine LIF

Nucleotide sequencing was performed by the dideoxy chain terminationmethod (Sanger et al, 1977) using the SEQUENASE (registered trade mark)reagents and protocol (United States Biochemicals). The nucleotidesequences of porcine and ovine LIF DNA are shown in FIGS. 2 and 3.Templates were single-stranded DNA of various fragments derived from the2.4 kbp BamHI fragment of pPLIFBaml or the 3 kbp BamHI fragment ofpOGLIFBaml subcloned into M13 phage vectors (Messing and Vieira 1982).The primers used were both an external primer in the M13 sequence and avariety of oligonucleotides complementary to sequences within the gene.

The porcine and ovine LIF sequences thus determined are shown in FIGS. 2and 3, respectively. Alignment of these sequences with the human andmouse gene sequences reveal that they contain coding regions specifyingproteins highly homologous to murine and human LIF. The protein sequenceencoded by these coding regions are listed above the nucleotidesequences.

The complete amino acid sequence of porcine and ovine LIF are alignedwith the murine and human LIF sequences in FIG. 4 with identitiesindicated by asterisks and conservative substitutions by dashes. Manylarge blocks of amino acid sequence remain totally conserved between allfour species. However, it is evident that the porcine sequence is moreclosely related to the ovine than the human and murine sequence. Acomparison of each of these four LIF sequences is presented in Table 1,in which only the mature portion of the LIF molecule is considered,excluding the hydrophobic leader. Only identities are scored in thiscomparison.

                  TABLE 1                                                         ______________________________________                                        Comparison of LIF amino acid sequences                                        (Percent Identity)                                                                   MURINE  HUMAN     OVINE     PORCINE                                    ______________________________________                                        MURINE:  100        78       74      77                                       HUMAN:             100       88      85                                       OVINE:                       100     83                                       PORCINE:                             100                                      ______________________________________                                    

The methods disclosed in PCT/AU88/00093 can be used for the constructionof a variety of expression vectors carrying the livestock (e.g. ovine orporcine) LIF gene. Such vectors include yeast (e.g. YEpsecl, Baldari etal, 1987), and E.coli e.g., vector pGEX-2T, Smith and Johnson, 1988Gearing et al, 1989;). Conditions for expression are as disclosed inPCT/AU88/00093.

EXAMPLE 5

The enhancement of the development of 8 cell murine embryos by additionof LIF is described in the following example, which is included by wayof illustration and not limitation of the present invention.

1. MATERIALS AND METHODS Animals

Balb-C×C57 three to four weeks old F1 female mice were primed with 7.5iu PMSG (Folligon; Intervet, Australia) followed 48 hours later, with7.5 iu hCG (Chorulon; Intervet, Australia) to achieve superovulation.Immediately following the hCG (Human chorionic Gonadotrophin) injection,treated females were placed with fertile males (CBA C57 strain, onefemale plus one male per cage). The next morning each female was checkedfor the presence of a vaginal plug as evidence of mating. This was thenconsidered as Day 1 of pregnancy.

Media

The culture medium was prepared from powdered Minimal Essential Medium(MEM; Eagle, with Earle's salts, with L-glutamine without sodiumbicarbonate; Flow Laboratories, UK) dissolved in Milli-Q water andsupplemented with 1 μg/ml glucose, 25 mM sodium bicarbonate and 10%(v/v) heat-inactivated fetal calf serum (FCS; CSL, Australia). Anantibiotic/antimycotic solution was also added to provide per 100 ml ofsolution, 10,000 units penicillin, 10,000 μg streptomycin and 25 μgfungizone (CSL, Australia). The pH and osmolarity of the media wereadjusted to 7.40 and 280 mOsm respectively. At this point the media wassterilised by filtration (Acrodisc 0.2 um filter; Gelman Sciences Inc.,USA).

Embryos

On Day 3 of pregnancy females were killed between 1300-1500 hours, i.e.71-73 hours post-hCG injection, by cervical dislocation. The wholereproductive tract was dissected out and placed in Earle's Balanced SaltSolution without Calcium and Magnesium (EBS9) at 37° C. Subsequently,8-cell embryos were teased/flushed out of the oviduct-uterus junctionand after washing once in culture medium were placed into control orexperimental group (see below) and maintained in a humidified gasenvironment of 5% CO₂ in air, at 37° C.

Culture of Embryos

For experimentation, 8-cell embryos were randomly assigned to a controlor experimental group with each group consisting of eight replicateswith embryos from four to six mice used per replicate. Embryos wereadded 10-20 per well approximately 15-20 minutes after recovery from theuterus and maintained in vitro for a period of five days in wellscontaining the culture media alone (1 ml/well) or the culture media withLIF (1000 u/ml) supplementation as indicated. This dosage of LIF waschosen as it is optimal for the inhibition of differentiation of EScells (International Patent Application No. PCT/AU89/00330).

Assessment of Morphological Development

Observations on embryo development were made daily using an invertedmicroscope and the numbers of embryos achieving morula, blastocyst orhatching blastocyst stage recorded (Hsu, 1979). On Days 4-5 of culture,many embryos underwent developmental changes associated withimplantation (Sherman, 1978). For this study, post hatching embryos wererecorded as achieving stage 1 when they displayed proliferatingtrophectoderm cells, and stage 2 when they showed outgrowth oftrophectoderm cells.

2. RESULTS

The effect on the development of the mouse 8-cell embryos in vitro ofincluding LIF (10³ units/ml in culture medium, prior, to (PRE) orfollowing (POST) formation of the blastocysts are shown in Table 2. Theresults are expressed as % initial number of embryos (n=35) completingthe developmental stage.

                  TABLE 2                                                         ______________________________________                                        LIF                                                                           PRE   POST    8-cell → BLASTOCYST → IMPLANTATION                ______________________________________                                        *-    -       100            57.6                                             +     -       100            67.2                                             -     +       100            85.7                                             +     +        99            77.2                                             Control only                                                                  ______________________________________                                    

By combining data on all experiments where LIF (10³ units/ml) has beenadded to the culture medium, a definite effect has been found where theaddition of LIF enhances the development of 8 cell mouse embryos to theimplantation stage 2 (see Materials and Methods--Assessment ofMorphological Development) as follows:

    ______________________________________                                                              Control  LIF                                            Embryos to Implantation Stage 2 =                                                                   226      156                                            ______________________________________                                        Total No. 8-cell Embryos Cultured                                                                   349      195                                                                  (64%)    (80%)                                          (χ.sup.2 = 27.0 P ≦ 0.001)                                         ______________________________________                                    

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

EXAMPLE 6 Expression of ovine LIF

A contiguous coding region for ovine LIF was constructed by intronremoval and site directed mutagenesis in a manner analogous to the humanLIF gene as previously described.

The ovine LIF coding region so constructed was cloned into yeastexpression vector YEpsec-1 in the correct (clones 3 and 15) andincorrect (clone 5) orientation. LIF activity was determined and theresults are shown in Table 3. LIF activity is expressed as Units/ml asdetermined using the M1 cell differentiation bioassay as describedbefore. The mouse positive control (mouse +ve) is a yeast clonecontaining YEpsec-1 with the murine LIF gene inserted in the correctorientation.

                  TABLE 3                                                         ______________________________________                                                      LIF Activity (Units)                                            Yeast Clone   (Units/ml)                                                      ______________________________________                                        Clone 3       10,700                                                          Clone 15      829,000                                                         Clone 5          0                                                            mouse + ve    61,400                                                          ______________________________________                                    

EXAMPLE 7 Receptor Binding Competition Assay

The receptor binding competition assay was performed as previouslydescribed. The assay shows the ability of yeast derived sheep LIF tocompete with iodinated murine LIF for binding to specific cellularreceptors on mouse liver cells.

                  TABLE 4                                                         ______________________________________                                                         [](ng/ml) .sup.125 I.LIF Specifically                        Competitor       bound cpm                                                    ______________________________________                                        pure rec. human (E. coli)                                                                      10000       0                                                                 1000        323                                                               100         575                                                               10          258                                                               1           1053                                                              0.1         1279                                                              0.01        1600                                             pure rec human LIF                                                                             100         614                                              (yeast)                                                                                        1           1078                                             crude sheep LIF (yeast)                                                                        1:1         625                                                               1:10        822                                              Saline           --          2549                                             untransf. yeast medium                                                                         1:1         2603                                                              1:10        2591                                             ______________________________________                                    

REFERENCES CITED

Baldari et al. EMBO J 6: 229-234, 1987

Hsu Y-C, Developmental Biology 68 4530616, 1979

Maniatis et al. Molecular Cloning. A Laboratory Manual. Cold SpringHarbor Laboratory, Cold Spring Harbor USA.

Messing and Vieira, Gene 19: 269-276, 1982

Sanger et al. Proc. Natl. Acad. Sci. USA 74: 5463-5462, 1977

Sherman, Methods in Mammalian Reproduction, New York: Academic Press pp81-125, 1978

Smith and Johnson Gene 67: 31-40, 1988

Gearing, Nicola, Metcalf, Foote, Willson, Gough and WilliamsBiotechnology 7: 1157-1161, 1989

We claim:
 1. A method for the in vitro development of a mammalian embryoto the implantation stage which method comprises the step of culturingthe embryo in vitro in a culture medium containing an effective amountof mammalian LIF and for a time and under conditions sufficient toincrease the percentage of eight cell embryos which reach theimplantation stage.
 2. The method according to claim 1 wherein themammalian embryo is of human, murine or livestock animal origin.
 3. Themethod according to claim 2 wherein the mammalian embryo is isolatedfrom a livestock species.
 4. The method according to claim 1 wherein themammalian LIF is from human, mouse or livestock species.
 5. The methodaccording to claim 4 wherein the mammalian LIF is from livestockspecies.
 6. The method according to claim 5 wherein the livestockspecies is a sheep, pig, goat, cow, horse or donkey.
 7. The methodaccording to claim 6 wherein the livestock species is a sheep or a pig.